Decellularization of Whole Human Liver Grafts Using Controlled Perfusion for Transplantable Organ Bioscaffolds

Abstract

Liver transplantation is the only effective treatment for end-stage liver disease, but absolute donor shortage remains a limiting factor. Recent advances in tissue engineering focus on generation of native extracellular matrix (ECM) by decellularized complete livers in animal models. Although proof of concept has been reported for human livers, this study aims to perform whole liver decellularization in a clinically relevant series using controlled machine perfusion. In this study, we describe a mild nondestructive decellularization protocol, effective in 11 discarded human whole liver grafts to generate constructs that reliably maintain hepatic architecture and ECM components using machine perfusion, while completely removing cellular DNA and RNA. The decellularization process preserved the ultrastructural ECM components confirmed by histology, electron microscopy, and proteomic analysis. Anatomical characteristics of the native microvascular network and biliary drainage of the liver were confirmed by contrast computed tomography scanning. Decellularized vascular matrix remained suitable for normal suturing and no major histocompatibility complex molecules were detected, suggesting absence of allo-reactivity when used for transplantation. After extensive washing, decellularized scaffolds were nontoxic for cells after reseeding human mesenchymal stromal or umbilical vein endothelial endothelium cells. Indeed, evidence of effective recellularization of the vascular lining was obtained. In conclusion, we established an effective method to generate clinically applicable liver scaffolds from human discarded whole liver grafts and show proof of concept that reseeding of normal human cells in the scaffold is feasible. This supports new opportunities for bioengineering of transplantable grafts in the future.

Strategies based on organ decellularization and recellularization

“…Various decellularization protocols have been established in mouse, rat, ferret and human-scale animals, such as sheep and pigs [11,54,69,[112][113][114]. Furthermore, the first protocols for decellularization of deceased human livers became available in 2017 in studies published by Verstegen et al [115].Nevertheless, no systematic comparisons of the damage different decellularization protocols exerts on the extracellular matrix or on recellularization results are available. Recently, researchers have focused not only on optimizing decellularization conditions through the surrounding oscillating pressure or controlling the pressure during the process but also on the reendothelialization and recellularization of whole livers in different preclinical trials.…”

A Hepatic Scaffold from Decellularized Liver Tissue: Food for Thought

“…An ideal synthetic scaffold must be a bioactive substrate capable of reproducing biophysical and biochemical characteristics of liver ECM. As reported above, synthetic scaffolds should provide a structural support function, promote cell viability and proliferation, and recreate an environment suitable for the diffusion of oxygen, nutrients, and cell growth factors [38]. Liver synthetic scaffolds may be manufactured from several types of biomaterials.…”

“…Later on, Verstegen et al [38] continued to exploit the use of marginal human livers, considered medically inappropriate for transplantation due to poor quality, as a prospective source of bioengineered hepatic scaffolding.…”

Reconstruction of structure and function in tissue engineering of solid organs: Toward simulation of natural development based on decellularization

“…Verstegen et al used dual machine perfusion to decellularize whole human livers. Perfusion fluid was delivered via the portal vein and the hepatic artery at controlled flow rates, leading to better preservation of functional ECM structure (Verstegen et al, ).…”